The major objective of the proposed studies is to further examine the concept that calcium flux plays an important role in initiating the actions of calcemic hormones on bone. We will utilize calcium-load liposomes to increase intracellular calcium. Liposome effects will be studied in the presence and absence of calcemic hormones (parathyroid hormone, prostaglandins, vitamin D metabolites). Hormonal effects on early events which may either a) initiate calcium translocation or b) produce rapid calcium-dependent changes will be investigated in bone organ cultures and bone cells. These will include investigations of the effects of calcemic hormones on phosphoinositol breakdown and on steady state levels of various phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol and polyphosphoinositides. Evidence for calcium-dependent kinases and calcium-dependent phosphorylation will be sought in bone; if these processes are present, we will examine their responses to calcemic hormones. In other studies we will continue to utilize the fetal rat bone organ culture system as a bioassay for vitamin D analogs and for 1.25-(OH)2vitamin D3 in serum and tissue extracts. A number of new metabolites and analogs of interest will be examined. The putative 1,25-(OH)2D which has been found to be produced by a number of cell cultures will be tested for biological activity on bone. We hope by these studies to gain a better understanding of the mechanisms of action of agents which are either etiologic factors in diseases of bone or potentially useful in their treatment.